T cells are a subset of white blood cells that contribute to diverse immune functions, including pathogen defense, autoimmunity, and cancer progression. Notably, recent advances in cancer therapy have indicated that T cells can be genetically modified to exhibit potent antitumor activity. Our ability to precisely engineer T cell with desired properties is lacking, however, due to insufficient tools and an incomplete understanding of the underlying molecular circuitry controlling T cell fate and function. The objective of this proposal is to use novel CRISPR (clustered regularly interspaced short palindromic repeat)-based genetic and transcriptional engineering of T cells to enhance cancer immunotherapy. The first half of the project will test whether deletion of immune checkpoint proteins will enhance function of chimeric antigen receptor (CAR) transduced T cells. The second half will screen for novel combinations of transcription factors that promote differentiation of memory CD8 T cells, a desirable subset of T cells for use in a type of cancer treatment known as adoptive transfer. Successful completion of this proposal will identify strategies for manipulating T cell fate and function in diverse clinical settings and should ultimately enhance development of vaccines and anticancer therapies.